12-oxophytodienoate reductase

Structure of 12-oxophytodienoate reductase (OPR3) bound to FMN cofactor, created in Pymol.^[1]

12-oxophytodienoate reductase (OPRs) is an enzyme of the family of Old Yellow Enzymes (OYE). ^[2] OPRs are grouped into two groups: OPRI and OPRII – the second group is the focus of this article, as the function of the first group is unknown, but is the subject of current research. ^[3] The OPR enzyme utilizes the cofactor flavin mononucleotide (FMN) and catalyzses the following reaction in the jasmonic acid synthesis pathway: ^[4]

Reaction catalyzed by 12-oxophytodienoate reductase in the jasmonic acid synthesis pathway.^[1]

This reaction occurs in peroxisomes in plants. ^[5] Several isozymes have been discovered, with varying substrate stereospecificity: three in Lycopersicon escultentum, 13 in Oryza sativa, and five in Arabidopsis thaliana. ^[6] The OPR3 isozyme is most extensively studied because it can reduce all 4 stereoisomers of the substrate, OPDA and because it has shown to be the most significant enzyme in the jasmonic acid synthesis pathway. ^[7][4]

Structure

12-oxophytodienoate reductase structure resembles OYE enzymes and has been elucidated by x-ray crystal structures. ^[1] The cDNA encodes 372 amino acids for this enzyme. ^[2] It exhibits a barrel fold of eight parallel beta-strands surrounded by eight alpha-helices to create a barrel shape. ^[6] Turns at the N-terminus loops of the beta-strands have been shown to contain three to four amino acid residues and the C-terminus loops range between three and 47 amino acid residues. ^[6] The C-terminus loops largely make up the active site and the larger range of the amount of residues is due to the diversity in the different isozyme active sites. ^[6]

OPR3, the most extensively studied isoform of 12-oxophytodienoate reductase, has a wider binding pocket than OPR1, which is enantioselective for only one OPDA substrate enantiomer. ^[1] The residues Tyr78 and Tyr246 that are at the mouth of the active site are responsible for the higher enantioselectivity of OPR1. ^{[8] [1]} OPR1 and OPR3 have identical substrate binding residues, but the difference in the width of the mouth of the active site determines the OPR1 specificity. ^[8][1]

12-oxophytodienoate reductase isomers OPR3 (blue) and OPR1 (green) with FMN cofactor (pink).^[1]

12-oxophytodienoate reductase has also been shown to practice self-inhibition by dimerization. ^[6] This is the only flavoprotein known to dimerize for inhibition and this dimerization is thought to be regulated by phosphorylation. ^[6] The dimerization occurs by the mutual binding of two loops into the two active sites. ^[6] These loops are highly evolutionarily conserved, indicating the dimerization is purposeful and significant in regulation. ^[6]

12-oxophytodienoate reductase (OPR3) dimerized to itself. This dimer provides self-inhibition of the enzyme.^[1]

Mechanism

The reduction mechanism employed has been shown to be a ping-pong, bi-bi mechanism. ^[6] The FMN cofactor is first reduced by NADPH, the substrate is then bound, and finally the substrate is reduced by a hydride transfer from NADPH to the substrate’s beta carbon. ^[6] The Km of OPR3 in Zea Mays was found to be 190 micromolar for its substrate OPDA. ^[9]

Biological Function

The reaction catalyzed by 12-oxophytodienoate reductase is in the jasmonic acid biosynthesis pathway. Jasmonic acid is known for its importance as a gene regulator for development and defense. ^{[4] [10] [11] [12]}

OPR3 is shown to be induced by touch, wind, UV light, application of detergent, wounding, and brassinosteroids. ^[4] In wound response, its activity has been shown to partially depend on jasmonic acid perception. ^[4] It is also shown to have greater enzyme efficiency than OPR1 and OPR2 in Arabidopsis Thaliana, showing it is the significant enzyme in the jasmonic acid biosynthesis pathway. ^[4]

Relevance to Agriculture

This enzyme is of interest in plant biology research because the disrupted OPR3 gene has been shown to cause male sterility in Arabidopsis Thaliana. ^[13] This is a point of interest in understanding the factors surrounding viable pollen development, a focus of research in the agriculture industry. ^[13]

Relevance to Phytoremediation

OPR has shown to also function in the reduction of explosive 2,4,6-trinitrotoluene (TNT). ^[14] Because TNT is a known toxic, environmental pollutant that is difficult to degrade, the use of phytoremediation to clean up sites contaminated with TNT is of significant interest. ^[14] OPR1 degraded TNT faster and with greater amount of degraded products than other isozymes. ^[14] This enzyme could therefore be used in phytoremediation. ^[14]

Phylogenetics

A phylogenetic analysis studying the structural evolution and functional divergence of the various OPR paralogues found seven conserved sub-families and suggested expansion of the OPR families occurred in land plants. ^[15] A total of 74 OPR genes in 11 species from six major plant lineages were found. ^[15] Surprisingly, introns were found to differ in length and number, but conserved in position, indicating successive intron loss. ^[15] The study also indicated that the substrate binding loop and the alpha-helices, but not the beta-sheets, were critical for functional divergence after sub-families were established and are therefore important in the OPR proteins. ^[15]

References